1. Field of the Invention
The present invention is directed to a method of derivatising an analyte for subsequent detection through a nucleic acid based sensor, and to a sensor based thereon.
2. Description of the Related Art
Since the introduction of in vitro evolution principles (Systematic Evolution of Ligands by Exponential enrichment, SELEX) in the late 1980, a vast number of synthetically derived nucleic acids have been established resembling a high specificity and affinity against a huge variety of target molecules. This class of molecules (hereinafter Functional Nucleic Acids, FNAs) recently has attracted much interest in fields like analysis for environmental monitoring, diagnosis, drug discovery and therapy to combat diseases.
In general sensors consist of two major units: a signal receptor unit and a signal transducing unit. Chemical receptor units comprise a synthetically derived material that interacts with the sample or the analyte. Low limits of detection are achieved by strong and direct chemical or physical interaction between the analyte and the receptor material. On the other hand biological receptor units comprising biological derived materials show a specific detection by the specific chemical or physical interaction between the biological receptor and the analyte molecule. The interaction between the receptor material and the analyte is measured as a change in various physical or chemical properties and is processed inside the transducing unit. This change is converted to a measureable signal correlating linear with the concentration of analyte molecules. It can be realized by changes of various properties like conductivity/resistivity, capacity, current, potential, light absorption and fluorescence.
Like antibodies or enzymes, FNAs have been used as highly specific biological receptor unit in bio-sensor applications against targets like peptides, proteins, DNA, RNA or organic and inorganic molecules. In principle, for FNA's exists no limitations concerning structure, size or status of the envisioned target molecule and one can evolve FNA's against analytes including those for which antibodies are difficult to obtain (metal ions, toxins or volatile compounds). Moreover, FNA based sensors can be established for demands where protein receptors are ineffectual (elevated temperature, non aqueous or complex environments).
FNA based sensors had shown in the past a remarkable selectivity. For example aptamers were found to selectively distinguish theophylline and caffeine by a factor of 10,000 where the only difference is a single methylene group inside the two molecules (U.S. Pat. No. 5,580,737). A modular designed FNA where the recognition domain is separated from the signal generating domain, allows more freedom in sensor design. Thereby a plurality of target molecules can be detected with the same sensing principle (electrical, optical, calorimetrical or gravimetrical). The de novo in vitro design of FNA based bio-sensor receptor units makes them exceedingly useful to determine and sense components out of complex environments with high specificity.
Beside their remarkably advantages, FNAs as a natural occurring material show also some major disadvantages. They are normally subjected to considerable degradation under physiological conditions which can be inhibited by capping or implementation of non-natural nucleotides like spiegelmere, fluorinated ribosyl residues and phosphorthioate. This makes them more resistive to hydrolytic or enzymatic degradation and potentially more useful for in vitro and in vivo sensor applications.
In general, for trace analysis of biological samples a signal amplification step is essential to increase the sensitivity. In case for FNAs as recognition element, DNAzymes proved to show a multiple turnover activity, which also can amplify readout signals. After activation through the analyte it can continuously activate previously inactive reporter molecules (e.g. stem-loop forming DNA).
This leads to a significant signal enhancement because each analyte molecule can create many reporter molecules.
Through these FNA properties in combination with transduction principles (electrical, optical, calorimetrical or gavimetrical) employing FNAs as recognition elements, peptides, proteins, DNA, RNA or organic and inorganic molecules were detected out of complex environments with high specificity and sensitivity.
A number of disadvantages and shortcomings limit the usage of FNAs as recognition element for sensing applications and trace analysis in complex environments for small, charged or nonpolar organic or inorganic molecules:
Target evaluation of FNAs is always performed using the SELEX process wherein a specific target molecule is immobilized onto a solid phase and a library of FNAs comprising a complexity of about 1014 randomly mutated molecules are used to select for a specific receptor-target interaction. However, recognition of target molecules dramaticaly worsen with the size and chemical nature of the target analyte making it almost impossible to get highly specific FNAs for small organic and inorganic compounds.
Another limitation is based on the fact that FNA can degrade rapidly under physiological condition. This degradation limits the time of operation of FNAs as the recognition unit in bio-sensors when operated under conditions where RNA/DNA can be hydrolyzed or is subjected to enzymatic decomposition. To overcome this shortcoming non natural nucleotides like spiegelmere, fluorinated ribosyl residues and phosphorthioates are commonly used. This implies the usage of complicated and expensive synthesis methodologies and the results do not meet the requirements necessary for selective and sensitive sensor applications.
In prior art, readout of DNAzyme amplified molecular beacon signal events occurs via fluorescent reporter molecules. This requires complex illumination, optics and detection systems, which therefore cannot easily be implemented into small devices.